A method for the production of phosphocreatine catalyzed by an immobilized enzyme

Abstract

The present application relates to the field of biological catalysis, and discloses a method for preparing creatine phosphate by immobilized enzyme catalysis, which comprises the following steps: mixing gelatin and a positive charge modifier to obtain a carrier liquid; adding creatine and magnesium salt to a crude creatine kinase solution for pre-incubation to realize conformational locking, then mixing with the carrier liquid and dropping into a low-temperature coagulation bath to solidify into gel microspheres, and then cross-linking and washing to obtain immobilized creatine kinase particles; finally, adding substrates, sodium hexametaphosphate, polyphosphate kinase and immobilized particles into a reaction system for reaction, and then separating and collecting the supernatant to obtain creatine phosphate. The present application protects the three-dimensional conformation of the active center of the enzyme by pre-incubation, uses the positive micro area of the carrier to electrostatically enrich the substrates to accelerate mass transfer, and introduces an auxiliary enzyme to construct an in-situ substrate regeneration system to re-phosphorylate the by-product adenosine diphosphate into adenosine triphosphate. The present application eliminates the feedback inhibition of the product, reduces the cost of raw materials, and improves the overall catalytic efficiency.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis technology, specifically to a method for preparing phosphocreatine using immobilized enzyme catalysis. Background Technology

[0002] Creatine phosphate is an important biochemical reagent and pharmaceutical intermediate. In industrial production, the synthesis of creatine phosphate from creatine using creatine kinase catalysis is a commonly used biotransformation pathway. This enzymatic catalytic process requires adenosine triphosphate (ATP) as a phosphate donor.

[0003] In existing single-enzyme free catalytic systems, the high market price of adenosine triphosphate (ATP), the reaction substrate, directly increases the production cost of creatine phosphate. As the catalytic reaction proceeds, a large amount of the byproduct adenosine diphosphate (ATP) is generated within the system. The accumulating ATP exerts feedback inhibition on creatine kinase, hindering the shift of the main reaction equilibrium towards the production of creatine phosphate, thus limiting the overall conversion rate.

[0004] To reduce costs and enable enzyme reuse, enzymes are typically immobilized using methods such as cross-linking. However, in traditional immobilization processes, the added cross-linking agents often fail to react with the enzyme surface groups. This reaction can easily affect the enzyme's active site, leading to physical blockage of catalytic sites or deformation of the enzyme molecule's three-dimensional conformation, resulting in a decrease in the initial activity of the immobilized enzyme. Furthermore, if the conventional carrier network structure is poorly designed, it will still face the problem of gradual enzyme leakage and decreased operational stability during subsequent long-term mechanically stirred reactions.

[0005] Immobilized enzymes constitute solid-liquid two-phase reaction systems in practical applications, exhibiting significant mass transfer resistance. The internal environment of traditional immobilized supports lacks the ability to enrich specific reaction substrates. Because substrate molecules such as adenosine triphosphate (ATP) carry a large number of negative charges, they diffuse randomly in the liquid phase solely based on their natural concentration gradient, making it difficult to form localized high-concentration aggregations within the immobilized support and around the enzyme molecules. This slow interfacial mass transfer rate limits the frequency of substrate contact between the immobilized enzyme and the substrate, hindering the improvement of the overall catalytic efficiency of the system. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for preparing phosphocreatine using immobilized enzyme catalysis. This method solves the problems of poor catalytic stability of existing creatine kinase in the free state and difficulty in recycling it after the reaction; in addition, the high cost of the reaction-dependent adenosine triphosphate (ATP) and the feedback inhibition of the generated byproduct adenosine diphosphate (DP), all of which contribute to the high cost of phosphocreatine preparation and the difficulty in improving the overall catalytic efficiency.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing phosphocreatine by immobilized enzyme catalysis, comprising:

[0008] Dissolve 5-15 parts by weight of gelatin in 99-110 parts by weight of deionized water, add 0.05-1.5 parts by weight of positive charge modifier, mix well and cool down to obtain a blended carrier solution for constructing positively charged microregions.

[0009] Add 0.1-0.5 parts by weight of creatine and 0.1-0.5 parts by weight of magnesium salt to 20-40 parts by weight of crude creatine kinase enzyme solution for pre-incubation to achieve conformational locking protection, and then mix with the blended carrier solution to obtain the gelation mixture. The gelling mixture was dropped into a coagulation bath containing surfactant at 4°C and solidified into gel microspheres. The gel microspheres were placed in a buffer solution containing 0.2-1.0 parts by weight of crosslinking agent for crosslinking and washing to obtain immobilized creatine kinase particles; Creatine, disodium adenosine triphosphate, magnesium salt, sodium hexametaphosphate, polyphosphokinase coenzyme solution, and the immobilized creatine kinase particles were added to the buffer system for reaction. The polyphosphokinase coenzyme solution and the sodium hexametaphosphate form an in-situ regeneration system to rephosphorylate the byproduct adenosine diphosphate to adenosine triphosphate. After the reaction, the immobilized creatine kinase particles are separated and the supernatant is collected to obtain phosphocreatine.

[0010] By adopting the above technical solution, the core principle of this invention is based on dual-enzyme coupling and enzyme conformation locking technology. Two key enzymatic processes exist in the entire reaction system: The main reaction is catalyzed by creatine kinase, with magnesium ions as a cofactor, to promote the reaction of creatine with adenosine triphosphate to produce phosphocreatine and adenosine diphosphate. In the side reaction, polyphosphokinase catalyzes the regeneration of the substrate, combining the adenosine diphosphate produced in the main reaction with inexpensive sodium hexametaphosphate, and converting it back into adenosine triphosphate and degraded sodium polyphosphate.

[0011] To ensure the efficient operation of this catalytic system, this invention specifically designs the immobilization microenvironment and the state of enzyme molecules. Before enzyme immobilization, cross-linking agents often cause irreversible damage to the enzyme's active site. Therefore, this method pre-introduces trace amounts of creatine and magnesium salts into the crude creatine kinase enzyme solution, allowing them to pre-enter the enzyme's active site and complete substrate occupancy. Because this occupancy maintains the original three-dimensional conformation of the catalytic site, even if a cross-linking agent is subsequently added, it can prevent excessive binding of cross-linking molecules and blockage of the catalytic site, thereby maximizing the preservation of the immobilized enzyme activity.

[0012] Meanwhile, the environment of the support itself also affects the catalytic efficiency. Considering that both the reaction substrate adenosine triphosphate (ATP) and the product creatine phosphate carry a significant negative charge in the liquid phase, this invention incorporates a positive charge modifier into gelatin as an immobilization framework. This blending method constructs positively charged microdomains within the gel, actively drawing the free negatively charged substrate into the microspheres through electrostatic attraction; the natural increase in local substrate concentration directly accelerates the mass transfer and reaction process.

[0013] Furthermore, the accumulation of byproducts often disrupts the continuity of the main reaction. Thanks to the aforementioned dual-enzyme coupling mechanism, the generated adenosine diphosphate (ATP) can be regenerated in situ into adenosine triphosphate (ATP) under the action of polyphosphokinase. This not only eliminates the feedback inhibition of ATP on creatine kinase, forcing the reaction equilibrium to continuously shift towards the target product phosphocreatine, but also significantly reduces the initial feed requirement for high-valent ATP. In addition, the immobilized particles themselves are easy to physically separate and recover, thus ensuring the economic efficiency of the entire process.

[0014] Preferably, the positive charge modifier is polyethyleneimine or chitosan oligosaccharide; the temperature control conditions for dissolving gelatin in deionized water are as follows: stir in a water bath at 50-60°C until dissolved, add the positive charge modifier and stir for 30-60 minutes, then lower the system temperature and maintain it at 35-40°C.

[0015] By employing the above technical solution, the selected polyethyleneimine or chitosan oligosaccharide has abundant amino groups on its molecular chain. These groups constitute dense positive charge sites, further amplifying the aforementioned electrostatic enrichment effect of positively charged micro-regions. Regarding temperature control in carrier preparation, the initial water bath at 50-60℃ allows the gelatin molecules to fully expand and dissolve; the subsequent cooling to 35-40℃ maintains the necessary fluidity of the mixture while avoiding the risk of thermal inactivation that can easily occur when directly contacting the enzyme solution.

[0016] Preferably, the coagulation bath containing surfactant is prepared by mixing 490-510 parts by weight of Tris-HCl buffer solution with a pH of 8.0-8.5 for coagulation with 1-3 parts by weight of polyoxyethylene sorbitan monooleate; the gelling mixture is dripped into the coagulation bath containing surfactant through a micro-injection pump at a flow rate of 1.0-2.0 ml per minute, and allowed to stand for aging for 30-60 minutes.

[0017] By employing the above technical solution, the polyoxyethylene sorbitan monooleate introduced into the coagulation bath weakens the surface tension at the droplet interface, allowing the mixture to be gelled to shrink into relatively uniform spheres upon droplet formation. When the droplets enter a low-temperature environment of 4°C, the gelatin macromolecular chains undergo a phase transition and solidify due to rapid physical entanglement upon cooling. The resulting porous network gel microspheres provide reliable structural support for the subsequent cross-linking reaction.

[0018] Preferably, the pre-incubation conditions are slow stirring and incubation at 20-30°C for 10-30 minutes; the cross-linking washing conditions are stirring and cross-linking at 4°C for 2-6 hours.

[0019] The reason for setting the pre-incubation in a mild range of 20-30℃ using the above technical solution is to allow the substrate molecules sufficient time and suitable kinetic conditions to accurately integrate into the enzyme's active pocket, thus achieving conformational locking. The subsequent cross-linking process is carried out at 4℃ mainly to deliberately suppress the reaction rate of the cross-linking agent; this slow cross-linking can prevent excessive network contraction from causing compression and distortion of the enzyme molecules encapsulated inside the microspheres, thereby physically stabilizing the enzyme's three-dimensional spatial morphology.

[0020] Preferably, the proportions and conditions for adding reactants to the buffer system are as follows: 2.5-10 parts by weight of creatine, 5-12 parts by weight of disodium adenosine triphosphate, 2.5-6 parts by weight of magnesium salt, 5-20 parts by weight of sodium hexametaphosphate, 10-30 parts by weight of polyphosphokinase coenzyme solution, and 15-50 parts by weight of immobilized creatine kinase particles; adjust the pH of the buffer system to 8.0-8.5, and react for 1-5 hours at 20-50°C with stirring. The magnesium salt is magnesium sulfate heptahydrate, and the cross-linking agent is glutaraldehyde.

[0021] By employing the above technical solution, a specific substrate-to-enzyme ratio establishes a dynamic balance between the consumption rate of adenosine triphosphate (ATP) in the main reaction and the regeneration rate of the coenzyme system. Simultaneously, maintaining a pH environment of 8.0-8.5 precisely covers the overlapping activity range of the two kinases. Glutaraldehyde, acting as a cross-linking agent, utilizes its dialdehyde structure to undergo a Schiff base reaction with gelatin molecules and free amino groups on the enzyme surface; this covalently linked, robust network is crucial to preventing significant leakage of enzyme molecules from the microsphere pores during prolonged stirring.

[0022] Preferably, the preparation process of crude creatine kinase enzyme solution includes: inoculating recombinant *E. coli* containing the rabbit creatine kinase gene into Luria-Bertani liquid medium and culturing it; adding an inducer to induce expression; collecting wet cells by centrifugation; suspending the wet cells in lysis buffer and homogenizing them using a high-pressure homogenizer to obtain cell lysate; incubating the cell lysate in a constant temperature water bath at 40-45℃ for 15-20 minutes to achieve thermal denaturation of endogenous contaminating enzymes in the host; then centrifuging to remove the precipitate and collecting the supernatant to obtain crude creatine kinase enzyme solution after thermal passivation and impurity removal. The inducer is isopropyl-β-D-thiogalactoside; the culture and induction process is as follows: inoculating recombinant *E. coli* into Luria-Bertani liquid medium and culturing it at 30-37℃ to the logarithmic phase; lowering the culture temperature to 16-30℃; adding 0.05-0.2 parts by weight of isopropyl-β-D-thiogalactoside; and continuing to culture for 16-24 hours. The process of suspending wet bacterial cells in lysis buffer is as follows: 9-11 parts by weight of wet bacterial cells are suspended in 50-100 parts by weight of Tris-HCl lysis buffer with a pH of 7.5-8.0, and the cells are cycled 2-4 times at a pressure of 600-1000 bar. The centrifugation conditions for removing the precipitate are: centrifugation at 8000-12000 rpm for 10-20 minutes.

[0023] By employing the above-described technical solution, this invention cleverly utilizes the physical difference that the recombinant rabbit creatine kinase exhibits superior thermostability compared to the endogenous proteins of the E. coli host, performing targeted, brief heat treatment at 40-45°C. Within this specific temperature range, the spatial structure of numerous host enzymes is disrupted, leading to aggregation and precipitation, while the target product, creatine kinase, remains well-dissolved. Combined with conventional centrifugation, this non-chromatographic purification method is not only rapid and inexpensive but also effectively removes impurity proteins that readily react with cross-linking agents or unnecessarily consume reaction substrates.

[0024] Preferably, the preparation process of the polyphosphokinase coenzyme solution includes: Recombinant Escherichia coli containing the Escherichia coli polyphosphokinase gene was inoculated into Luria-Bertani liquid medium and cultured. After inducing expression with an inducer, the polyphosphokinase wet cells were collected by centrifugation. The polyphosphokinase wet cells were suspended in Tris-HCl buffer for lysis and lysed to obtain polyphosphokinase cell lysate. The polyphosphokinase cell lysate was centrifuged at 4°C to remove the precipitate, and the supernatant was collected to obtain polyphosphokinase coenzyme solution.

[0025] By employing the above technical solution and through specific induction expression and centrifugation processes, an auxiliary biocatalyst capable of degrading polyphosphates and completing phosphate group transfer was prepared. This essentially completes the necessary enzyme source puzzle for the aforementioned substrate in-situ regeneration system, ensuring the smooth operation of the entire dual-enzyme coupling circuit.

[0026] This invention provides a method for preparing phosphocreatine using immobilized enzyme catalysis. It has the following beneficial effects: 1. In this invention, creatine kinase is pre-incubated with creatine and magnesium salts before immobilization and cross-linking, allowing the substrate to enter the enzyme active site in advance and form a site, thus maintaining the three-dimensional conformation of the enzyme at the physical level. This avoids the reactive groups from blocking the catalytic site when cross-linking agents are added later, thereby preserving a high initial enzyme activity during the immobilization process. The formed cross-linking network prevents the leakage of enzyme molecules during long-term stirring reactions, improving the reusability and operational stability of the enzyme.

[0027] 2. This invention constructs positively charged microregions within a gel carrier by blending positively charged modifiers such as polyethyleneimine or chitosan oligosaccharide with gelatin. Since substrate molecules such as adenosine triphosphate in the reaction system carry negative charges, the positively charged environment inside the carrier can enrich the substrate in the liquid phase using electrostatic attraction, thereby increasing the local substrate concentration around the immobilized enzyme, accelerating the mass transfer rate between the liquid and solid phases, and improving the overall catalytic efficiency of the system.

[0028] 3. This invention introduces polyphosphokinase and sodium hexametaphosphate into the reaction system to construct an in-situ regeneration system, which can rephosphorylate adenosine diphosphate (ATP) produced by the main reaction into adenosine triphosphate (ATP), thereby relieving the feedback inhibition of ATP on creatine kinase, promoting the main reaction equilibrium to shift towards the production of phosphocreatine, and reducing the initial feed requirement of high-valent ATP, thus lowering the preparation cost of phosphocreatine. Attached Figure Description

[0029] Figure 1 The diagram shows the comparison of enzyme activity under different crude enzyme solution treatment methods of the present invention, wherein (a) is the comparison of target creatine kinase enzyme activity, and (b) is the comparison of endogenous enzyme activity. Figure 2 The double reciprocal plot of the enzymatic reactions under different immobilized carrier microenvironments in this invention is shown. Figure 3 This is a diagram showing the evolution of enzyme activity retention under different immobilization processes according to the present invention; Figure 4 The diagram shows the comparison of the final yield of the reaction under different catalytic systems of the present invention. (a) is the absolute concentration of phosphocreatine generated in the system at the end of the reaction of each test group, and (b) is the molar conversion diagram of creatine based on substrate consumption. Figure 5This is a graph showing the change in relative enzyme activity retention rate under multiple batches of cyclic catalysis according to the present invention. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Preparation Examples 1-4: Preparation Example 1: This preparation example provides a method for preparing crude creatine kinase solution, including the following steps: Recombinant *E. coli* BL21(DE3) containing the rabbit creatine kinase gene was inoculated into 1000 parts by weight of Luria-Bertani (LB) liquid medium and cultured at 35°C to the logarithmic growth phase. The culture temperature was then lowered to 25°C, and 0.1 parts by weight of isopropyl-β-D-thiogalactoside was added. Culture was continued for 20 hours. Ten parts by weight of wet cells were collected by centrifugation and resuspended in 50 parts by weight of Tris-HCl buffer (pH 7.8). The cells were homogenized three times at 800 bar using a high-pressure homogenizer to obtain cell lysate. The cell lysate was incubated in a 42°C water bath for 18 minutes, followed by centrifugation at 10,000 rpm for 15 minutes. The precipitate was removed, and the supernatant was collected as the crude creatine kinase enzyme solution.

[0032] Preparation Example 2: This preparation example provides a method for preparing crude creatine kinase solution, including the following steps: Recombinant *E. coli* BL21(DE3) containing the rabbit creatine kinase gene was inoculated into 1000 parts by weight of LB liquid medium and cultured at 30°C to the logarithmic growth phase. The culture temperature was then lowered to 16°C, and 0.05 parts by weight of isopropyl-β-D-thiogalactoside was added. Culture was continued for 16 hours. Ten parts by weight of wet cells were collected by centrifugation and resuspended in 50 parts by weight of Tris-HCl buffer (pH 7.5). The cells were homogenized twice using a high-pressure homogenizer at 600 bar to obtain cell lysate. The cell lysate was incubated in a 40°C water bath for 15 minutes, followed by centrifugation at 8000 rpm for 10 minutes. The precipitate was removed, and the supernatant was collected as the crude creatine kinase enzyme solution.

[0033] Preparation Example 3: This preparation example provides a method for preparing crude creatine kinase solution, including the following steps: Recombinant *E. coli* BL21(DE3) containing the rabbit creatine kinase gene was inoculated into 1000 parts by weight of LB liquid medium and cultured at 37°C to the logarithmic growth phase. The culture temperature was then lowered to 30°C, and 0.2 parts by weight of isopropyl-β-D-thiogalactoside was added. Culture was continued for 24 hours. Ten parts by weight of wet cells were collected by centrifugation and resuspended in 100 parts by weight of Tris-HCl buffer (pH 8.0). The cells were homogenized four times at 1000 bar using a high-pressure homogenizer to obtain cell lysate. The cell lysate was incubated in a 45°C water bath for 20 minutes, followed by centrifugation at 12000 rpm for 20 minutes. The precipitate was removed, and the supernatant was collected as the crude creatine kinase enzyme solution.

[0034] Preparation Example 4: This preparation example provides a method for preparing polyphosphokinase coenzyme solution, including the following steps: Recombinant *E. coli* BL21(DE3) containing the *E. coli* polyphosphokinase* gene was inoculated into 1000 parts by weight of LB liquid medium and cultured at 35°C to the logarithmic growth phase. The culture temperature was then lowered to 25°C, and 0.1 parts by weight of isopropyl-β-D-thiogalactoside was added. Culture was continued for 20 hours. Ten parts by weight of wet cells were collected by centrifugation and resuspended in 50 parts by weight of Tris-HCl buffer (pH 7.8). The cells were homogenized three times using a high-pressure homogenizer at 800 bar to obtain cell lysate. The cell lysate was centrifuged at 10,000 rpm at 4°C for 15 minutes to remove the precipitate. The supernatant was collected as the polyphosphokinase coenzyme solution.

[0035] Examples 1-4: Example 1: This embodiment provides a method for preparing phosphocreatine using immobilized enzyme catalysis, comprising the following steps: (1) Preparation of blended carrier liquid: Add 10 parts by weight of gelatin to 100 parts by weight of deionized water, stir in a water bath at 55°C until completely dissolved, slowly add 0.5 parts by weight of polyethyleneimine, stir thoroughly for 45 minutes, then lower the system temperature and maintain it at 38°C to obtain a liquid blended carrier liquid.

[0036] (2) Conformation locking and mixing: Take 30 parts by weight of the crude creatine kinase enzyme solution prepared in Preparation Example 1, add 0.2 parts by weight of creatine and 0.2 parts by weight of magnesium sulfate heptahydrate to it, and slowly stir and incubate at 25°C for 20 minutes. Then mix the pre-incubated enzyme solution with the above-mentioned blended carrier solution at 38°C to obtain the gelation mixture.

[0037] (3) Physical condensation granulation: 500 parts by weight of Tris-HCl buffer solution with a pH of 8.2 and 2 parts by weight of polyoxyethylene sorbitan monooleate were mixed and pre-cooled to 4°C as a coagulation bath. Using a micro-injection pump, the above gelling mixture was dripped uniformly into the low-temperature coagulation bath at a flow rate of 1.5 ml per minute through a syringe needle with an inner diameter of 0.5 mm. The gelatin droplets rapidly underwent a physical phase change and solidified upon cooling, forming gel microspheres. The microspheres were then allowed to stand and age in the coagulation bath for 45 minutes to strengthen the structure.

[0038] (4) Mild chemical crosslinking: The gel microspheres were filtered out and transferred to 200 parts by weight of buffer solution containing 0.5 parts by weight of glutaraldehyde. The microspheres were then gently stirred at 4°C for 4 hours for crosslinking. After crosslinking, the microspheres were washed repeatedly with fresh buffer solution 4 times to remove residual crosslinking agent, resulting in microenvironmentally charged immobilized creatine kinase particles.

[0039] (5) Enzymatic synthesis reaction: 1000 parts by weight of buffer solution was added to a reactor equipped with temperature-controlled stirring, followed by 5 parts by weight of creatine, 10 parts by weight of disodium adenosine triphosphate, 5 parts by weight of magnesium sulfate heptahydrate, 10 parts by weight of sodium hexametaphosphate, and 20 parts by weight of the polyphosphokinase coenzyme solution prepared in Preparation Example 4. The pH of the system was adjusted to 8.2. Then, 30 parts by weight of the above-mentioned immobilized creatine kinase particles were added to the system. The reaction was carried out at 35°C and a stirring speed of 200 rpm for 3 hours. After the reaction was completed, the immobilized enzyme particles were separated through a filter screen, and the supernatant reaction solution was collected to obtain a product solution containing phosphocreatine.

[0040] Example 2: This embodiment provides a method for preparing phosphocreatine using immobilized enzyme catalysis, comprising the following steps: (1) Preparation of blended carrier liquid: Add 5 parts by weight of gelatin to 100 parts by weight of deionized water, stir in a water bath at 50°C until completely dissolved, slowly add 0.05 parts by weight of polyethyleneimine, stir thoroughly for 30 minutes, then lower the system temperature and maintain it at 35°C to obtain a liquid blended carrier liquid.

[0041] (2) Conformation locking and mixing: Take 20 parts by weight of the crude creatine kinase enzyme solution prepared in Preparation Example 2, add 0.1 parts by weight of creatine and 0.1 parts by weight of magnesium sulfate heptahydrate to it, and slowly stir and incubate at 20°C for 10 minutes. Then mix the pre-incubated enzyme solution with the above-mentioned blended carrier solution at 35°C to obtain the gelation mixture.

[0042] (3) Physical condensation granulation: 500 parts by weight of Tris-HCl buffer solution with a pH of 8.0 and 1 part by weight of polyoxyethylene sorbitan monooleate were mixed and pre-cooled to 4°C as a coagulation bath. Using a micro-injection pump, the above gelling mixture was dripped uniformly into the low-temperature coagulation bath at a flow rate of 1.0 ml per minute through a syringe needle with an inner diameter of 0.4 mm. The gelatin droplets rapidly underwent a physical phase change and solidified upon cooling, forming gel microspheres. The microspheres were then allowed to stand and age in the coagulation bath for 30 minutes to strengthen the structure.

[0043] (4) Mild chemical crosslinking: The gel microspheres were filtered out and transferred to 200 parts by weight of buffer solution containing 0.2 parts by weight of glutaraldehyde. The microspheres were then gently stirred at 4°C for 2 hours for crosslinking. After crosslinking, the microspheres were washed three times with fresh buffer solution to remove residual crosslinking agent, resulting in immobilized creatine kinase particles with microenvironment charge modification.

[0044] (5) Enzymatic synthesis reaction: 1000 parts by weight of buffer solution was added to a reactor equipped with temperature-controlled stirring, followed by 2.5 parts by weight of creatine, 5 parts by weight of disodium adenosine triphosphate, 2.5 parts by weight of magnesium sulfate heptahydrate, 5 parts by weight of sodium hexametaphosphate, and 10 parts by weight of the polyphosphokinase coenzyme solution prepared in Preparation Example 4. The pH of the system was adjusted to 8.0. Then, 15 parts by weight of the above-mentioned immobilized creatine kinase particles were added to the system. The reaction was carried out at 20°C and a stirring speed of 150 rpm for 1 hour. After the reaction was completed, the immobilized enzyme particles were separated through a filter screen, and the supernatant reaction solution was collected to obtain a product solution containing phosphocreatine.

[0045] Example 3: This embodiment provides a method for preparing phosphocreatine using immobilized enzyme catalysis, comprising the following steps: (1) Preparation of blended carrier liquid: Add 15 parts by weight of gelatin to 100 parts by weight of deionized water, stir in a water bath at 60°C until completely dissolved, slowly add 1.5 parts by weight of polyethyleneimine, stir thoroughly for 60 minutes, then lower the system temperature and maintain it at 40°C to obtain a liquid blended carrier liquid.

[0046] (2) Conformation locking and mixing: Take 40 parts by weight of the crude creatine kinase enzyme solution prepared in Preparation Example 3, add 0.5 parts by weight of creatine and 0.5 parts by weight of magnesium sulfate heptahydrate to it, and slowly stir and incubate at 30°C for 30 minutes. Then mix the pre-incubated enzyme solution with the above-mentioned blended carrier solution at 40°C to obtain the gelation mixture.

[0047] (3) Physical condensation granulation: 500 parts by weight of Tris-HCl buffer solution with a pH of 8.5 and 3 parts by weight of polyoxyethylene sorbitan monooleate were mixed and pre-cooled to 4°C as a coagulation bath. Using a micro-injection pump, the above gelling mixture was dripped uniformly into the low-temperature coagulation bath at a flow rate of 2.0 ml per minute through a syringe needle with an inner diameter of 0.8 mm. The gelatin droplets rapidly underwent a physical phase change and solidified upon cooling, forming gel microspheres. The microspheres were then allowed to stand and age in the coagulation bath for 60 minutes to strengthen the structure.

[0048] (4) Mild chemical crosslinking: The gel microspheres were filtered out and transferred to 200 parts by weight of buffer solution containing 1.0 part by weight of glutaraldehyde. The microspheres were then gently stirred at 4°C for 6 hours for crosslinking. After crosslinking, the microspheres were washed repeatedly with fresh buffer solution 5 times to remove residual crosslinking agent, resulting in microenvironmentally charged immobilized creatine kinase particles.

[0049] (5) Enzymatic synthesis reaction: 1000 parts by weight of buffer solution were added to a reactor equipped with temperature-controlled stirring, followed by the sequential addition of 10 parts by weight of creatine, 12 parts by weight of disodium adenosine triphosphate, 6 parts by weight of magnesium sulfate heptahydrate, 20 parts by weight of sodium hexametaphosphate, and 30 parts by weight of the polyphosphokinase coenzyme solution prepared in Preparation Example 4. The pH of the system was adjusted to 8.5. Subsequently, 50 parts by weight of the above-mentioned immobilized creatine kinase particles were added to the system. The reaction was carried out at 50°C and a stirring speed of 250 rpm for 5 hours. After the reaction was completed, the immobilized enzyme particles were separated through a filter screen, and the supernatant reaction solution was collected to obtain a product solution containing phosphocreatine.

[0050] Example 4: This embodiment provides a method for preparing phosphocreatine using immobilized enzyme catalysis, comprising the following steps: (1) Preparation of blended carrier liquid: Add 10 parts by weight of gelatin to 100 parts by weight of deionized water, stir in a water bath at 55°C until completely dissolved, slowly add 0.5 parts by weight of chitosan oligosaccharide, stir thoroughly for 45 minutes, then lower the system temperature and maintain it at 38°C to obtain a liquid blended carrier liquid.

[0051] (2) Conformation locking and mixing: Take 30 parts by weight of the crude creatine kinase enzyme solution prepared in Preparation Example 1, add 0.2 parts by weight of creatine and 0.2 parts by weight of magnesium sulfate heptahydrate to it, and slowly stir and incubate at 25°C for 20 minutes. Then mix the pre-incubated enzyme solution with the above-mentioned blended carrier solution at 38°C to obtain the gelation mixture.

[0052] (3) Physical condensation granulation: 500 parts by weight of Tris-HCl buffer solution with a pH of 8.2 and 2 parts by weight of polyoxyethylene sorbitan monooleate were mixed and pre-cooled to 4°C as a coagulation bath. Using a micro-injection pump, the above gelling mixture was dripped uniformly into the low-temperature coagulation bath at a flow rate of 1.5 ml per minute through a syringe needle with an inner diameter of 0.5 mm. The gelatin droplets rapidly underwent a physical phase change and solidified upon cooling, forming gel microspheres. The microspheres were then allowed to stand and age in the coagulation bath for 45 minutes to strengthen the structure.

[0053] (4) Mild chemical crosslinking: The gel microspheres were filtered out and transferred to 200 parts by weight of buffer solution containing 0.5 parts by weight of glutaraldehyde. The microspheres were then gently stirred at 4°C for 4 hours for crosslinking. After crosslinking, the microspheres were washed repeatedly with fresh buffer solution 4 times to remove residual crosslinking agent, resulting in microenvironmentally charged immobilized creatine kinase particles.

[0054] (5) Enzymatic synthesis reaction: 1000 parts by weight of buffer solution was added to a reactor equipped with temperature-controlled stirring, followed by 5 parts by weight of creatine, 10 parts by weight of disodium adenosine triphosphate, 5 parts by weight of magnesium sulfate heptahydrate, 10 parts by weight of sodium hexametaphosphate, and 20 parts by weight of the polyphosphokinase coenzyme solution prepared in Preparation Example 4. The pH of the system was adjusted to 8.2. Then, 30 parts by weight of the above-mentioned immobilized creatine kinase particles were added to the system. The reaction was carried out at 35°C and a stirring speed of 200 rpm for 3 hours. After the reaction was completed, the immobilized enzyme particles were separated through a filter screen, and the supernatant reaction solution was collected to obtain a product solution containing phosphocreatine.

[0055] Comparative Examples 1-6: Comparative Example 1: Compared with Example 1, the difference is that no immobilization treatment was performed. Instead, the crude enzyme solution of free creatine kinase prepared in Example 1 with an equal amount of enzyme activity was directly added to the reaction system for catalytic reaction. All other aspects are the same.

[0056] Comparative Example 2: Compared with Example 1, the difference is that a traditional direct encapsulation crosslinking process is used. That is, polyethyleneimine is not added in step (1); creatine and magnesium sulfate heptahydrate are not added for conformational locking pre-incubation in step (2); and 0.5 parts by weight of glutaraldehyde are directly added to the mixture of crude enzyme solution and gelatin for crosslinking, without physical condensation granulation step, and the rest are the same.

[0057] Comparative Example 3: Compared with Example 1, the difference is that polyethyleneimine was not added in the preparation of the blended carrier liquid in step (1), but the rest are the same.

[0058] Comparative Example 4: Compared with Example 1, the difference is that in step (2), creatine and magnesium sulfate heptahydrate were not added in advance for conformation locking pre-incubation. Instead, the crude creatine kinase enzyme solution was directly mixed with the co-mixed carrier solution. All other aspects are the same.

[0059] Comparative Example 5: Compared with Example 1, the difference is that the crude creatine kinase enzyme solution used in step (2) was not subjected to 42°C constant temperature water bath heat passivation treatment during the preparation process, while the rest are the same.

[0060] Comparative Example 6: Compared with Example 1, the difference is that sodium hexametaphosphate and polyphosphokinase co-enzyme solution were not added to the enzymatic synthesis reaction system in step (5), but the rest are the same.

[0061] Test Examples 1-5: Test Example 1: This test case aims to verify the effect of heat treatment during the preparation of crude enzyme solution on the scavenging of endogenous interfering enzymes in host cells. It should be noted that, for ease of quantification and standardized calculation of experimental parameters, 1 part by weight in each preparation example and embodiment of this invention is uniformly converted to 1 gram (g) in this test case and subsequent test cases. Correspondingly, when liquid volume is involved, it is equivalently measured in milliliters (mL) or liters (L) based on its density characteristics. The specific experimental steps are as follows: The crude enzyme solution prepared in Example 1 after being kept at a constant temperature of 42°C in a water bath, and the cell lysate supernatant collected by centrifugation without heat passivation during the preparation of Comparative Example 5, were taken as test subjects.

[0062] The total protein concentration of the two groups of samples was determined by the Coomassie brilliant blue protein determination method. In the experiment, a standard curve was plotted using bovine serum albumin solutions with known gradients. The absorbance value at 595 nm was measured and substituted into the curve equation to calculate the true protein concentration of each sample.

[0063] The catalytic activity of the target creatine kinase was quantitatively determined. A Tris-HCl buffer substrate system containing a quantitative amount of creatine, disodium adenosine triphosphate (ATP), and magnesium sulfate heptahydrate was prepared in a reaction tube. An equal volume of the enzyme solution was added, and the reaction was carried out in a shaker at 35°C for 15 minutes. The reaction was terminated by adding trichloroacetic acid to a final mass fraction of 5% to 10%. The concentration of creatine phosphate generated in the supernatant was determined by high-performance liquid chromatography (HPLC). The amount of enzyme consumed to catalyze the production of 1 micromole of creatine phosphate per minute was defined as one standard creatine kinase activity unit. The specific enzyme activity of the target enzyme was calculated by combining this with the total protein concentration.

[0064] The residual activity of endogenous contaminants (mainly adenosine triphosphate hydrolases and nonspecific phosphatases) in the system was evaluated. A Tris-HCl buffer containing a final concentration of 5 mmol / L adenosine triphosphate was constructed as the test substrate. An equal volume of the enzyme solution was added, and the mixture was incubated at the same temperature. After the reaction, the concentration of free inorganic phosphate released in the system was determined using the molybdenum blue colorimetric method with ammonium molybdate and ascorbic acid at a wavelength of 660 nm. The amount of enzyme required to produce 1 μmol of inorganic phosphate per minute through nonspecific hydrolysis was defined as one unit of contaminant activity, and the specific enzyme activity was calculated accordingly.

[0065] Table 1. Specific enzyme activity test data of crude enzyme solution at different treatment stages

[0066] According to Table 1 and Figure 1 The data in (a) and (b) show that the total protein concentration of the crude enzyme solution used in Comparative Example 5, in its original state without heat treatment, was 14.83 mg / mL, with a background activity of 11.27 U / mg for endogenous contaminants. After treatment in a 42°C constant temperature water bath, the total protein concentration of the crude enzyme solution in Example 1 decreased to 7.21 mg / mL. In the biochemical separation and purification experiment, the significant decrease in the liquid phase protein concentration indicated that some host contaminants had undergone thermal denaturation and precipitation. The observed specific enzyme activity of endogenous contaminants decreased significantly to 0.58 U / mg, while the specific enzyme activity of the target creatine kinase increased to 68.94 U / mg due to the removal of contaminants and their increased weight.

[0067] Creatine kinase derived from rabbit muscle exhibits good thermal stability, while adenosine triphosphate (ATP) hydrolases, widely present in E. coli host cells, are highly sensitive to temperature changes. Under a predetermined incubation condition, host proteins aggregate into insoluble substances due to thermal denaturation, which are then effectively separated by high-speed centrifugation, thus eliminating interfering enzymes that can trigger side reactions. If these enzymes are not removed, their hydrolytic degradation of ATP will disrupt the energy balance of the substrate regeneration network, leading to premature termination of the main reaction due to substrate loss. This heating-based purification process effectively reduces the interference of endogenous enzyme activity without relying on chromatography resin, ensuring the smooth construction of subsequent substrate reaction pathways.

[0068] Test Example 2: This test case aims to verify the actual effect of introducing polyethyleneimine modification into the support on the mass transfer and affinity of adenosine triphosphate substrate by measuring enzymatic reaction kinetic parameters. The specific experimental steps are as follows: Immobilized creatine kinase particles containing polyethyleneimine modified prepared in Example 1 and immobilized creatine kinase particles without polyethyleneimine prepared in Comparative Example 3 were weighed out as test subjects and equilibrated and washed with reaction buffer before use.

[0069] Test buffer was added to a series of isothermal reactors to fix the creatine concentration at 50 mmol / L and maintain an oversaturated state. Magnesium sulfate heptahydrate was added to maintain a constant molar ratio of magnesium ions to adenosine triphosphate (ATP) of 1:1. Subsequently, a gradient concentration of ATP (specifically 1.0, 2.0, 3.0, 5.0, 8.0, and 12.0 mmol / L) was set, and the pH of the system was strictly controlled at 8.2.

[0070] Table 2. Initial reaction rate data of different immobilized particles at gradient substrate concentrations

[0071] According to Table 2 and Figure 2 The data from Example 1 and Comparative Example 3 show different catalytic kinetic parameters at different substrate concentrations. In solid-liquid multiphase catalytic systems, the bulk substrate concentration measured by liquid-phase sampling is usually higher than the concentration actually in contact with enzyme molecules inside the support. The measured apparent Michaelis constant is a comprehensive reflection of the enzyme's intrinsic affinity and the diffusion mass transfer efficiency of the support pores. Linvifer-Burke double reciprocal plotting of the extracted initial rate data reveals that the linear fitting line corresponding to the unmodified Comparative Example 3 has a smaller intercept on the horizontal axis, and the calculated apparent Michaelis constant is approximately 4.87 mmol / L.

[0072] In a slightly alkaline buffer solution, the isoelectric point characteristics of the gelatin carrier surface result in its backbone carrying a net negative charge, leading to electrostatic repulsion of adenosine triphosphate (ATP), which is also a polyanion. Example 1 used a crosslinking process of polyethyleneimine and gelatin. The protonated amine groups of polyethyleneimine neutralized the original negative charge of the carrier, reducing the electrostatic repulsion. The calculated apparent Michaelis constant decreased to approximately 1.60 mmol / L. The changes in kinetic parameters indicate that microenvironment charge regulation improved the catalytic efficiency of the immobilized enzyme for negatively charged substrates.

[0073] Test Example 3: This test case aims to investigate the protective effects of vector encapsulation methods and conformation-locking pretreatment on the active site of creatine kinase. The specific experimental steps are as follows: The crude creatine kinase enzyme solution obtained in Preparation Example 1 was extracted and its initial total enzyme activity in the free state was determined by Coomassie Brilliant Blue method combined with liquid chromatography substrate quantification at 35°C and pH 8.2. Subsequently, based on the principle of feeding materials with equal free total enzyme activity, immobilized creatine kinase particles were prepared as test objects according to the process flow of Example 1, Comparative Example 2, and Comparative Example 4, respectively. After preparation, all particles were thoroughly washed with buffer solution at pH 8.2 to remove unbound free enzyme and residual reagents.

[0074] After washing, the immobilized particles in each group were filtered dry, weighed, and then placed into reaction flasks containing 100 mL of standard test substrate solution. This test substrate solution contained 50 mmol / L creatine, and 10 mmol / L each of adenosine triphosphate disodium salt and magnesium sulfate heptahydrate.

[0075] The reaction flask was placed in a constant-temperature shaker and the catalytic reaction was started at 35°C and 200 rpm. Within 10 minutes of the reaction system reaching the linear initial velocity stage, samples were taken periodically using a pipette, and the samples were immediately injected into centrifuge tubes containing 5% trichloroacetic acid to quench the reaction.

[0076] The quenched sample was centrifuged at 12,000 rpm for 10 minutes. The supernatant was taken and the total amount of phosphocreatine generated was quantitatively determined by high performance liquid chromatography. The apparent total enzyme activity of each immobilized particle was calculated and compared with the total free enzyme activity recorded in step 1 to calculate the enzyme activity recovery rate under each process condition.

[0077] Table 3. Initial enzyme activity recovery data of creatine kinase under different immobilization processes

[0078] According to Table 3 and Figure 3 The data shows that the immobilization process has a significant impact on the retention of creatine kinase's catalytic activity. While chemical cross-linking agents improve the mechanical strength of the carrier, they usually lead to a significant loss of enzyme activity, as observed in the direct embedding cross-linking process used in Comparative Example 2. When glutaraldehyde solution is directly mixed with the free enzyme, the cross-linking agent easily undergoes a non-specific reaction with the active groups on the enzyme molecule surface, resulting in a recovery rate of only 18.60%, indicating that the catalytic centers of most enzyme molecules are damaged. Although Comparative Example 4 changed to low-temperature gelation followed by low-concentration soaking and cross-linking, its enzyme activity recovery rate was 42.42%, still at a low level. Example 1 added a pre-incubation step of creatine and magnesium ions before mixing the enzyme solution with the carrier, utilizing the substrate binding to occupy the space of the enzyme's active center. After the enzyme solution with pre-incubation protection was immobilized, the cross-linking reaction was mainly limited to the area between the amino groups on the outer periphery of the enzyme protein and the carrier backbone. The recovery rate of 84.21% indicates that substrate pre-binding effectively reduced the damage of chemical cross-linking agents to the enzyme's active center and preserved the catalytic site.

[0079] Test Example 4: This test case aims to verify the actual impact of the dual-enzyme coupled regeneration system and the entire scheme on the final reaction conversion rate. By comparing the final product concentrations of different reaction systems, the driving effect of the in-situ substrate regeneration mechanism on thermodynamic equilibrium is evaluated. It should be noted that in this test case and subsequent test cases, the activity unit (U) of the polyphosphokinase coenzyme solution is defined as: the amount of polyphosphokinase required to catalyze the generation of 1 micromole of adenosine triphosphate per minute in a buffer system at 35°C and pH 8.2, using sodium hexametaphosphate and adenosine diphosphate as substrates. The specific experimental steps are as follows: Prepare the basic catalytic reaction solution. Add a buffer solution with a pH of 8.2 to multiple reactors equipped with temperature control and magnetic stirring. Add creatine sequentially to achieve a final concentration of 50 mmol / L, then add disodium adenosine triphosphate and magnesium sulfate heptahydrate to achieve final concentrations of 5 mmol / L for each. For the test groups of Examples 1 to 4 and Comparative Example 5, an additional sodium hexametaphosphate at a final concentration of 60 mmol / L was added as a free phosphate donor; Comparative Example 6, as a control without regeneration, did not include sodium hexametaphosphate.

[0080] Immobilized creatine kinase particles corresponding to Examples 1 to 4, Comparative Examples 5 and 6, with an initial total enzyme activity of 1000 U (activity units), were added to each reactor. Subsequently, polyphosphokinase coenzyme solution with a total enzyme activity of 500 U, prepared in Preparation Example 4, was added to the reaction systems other than Comparative Example 6 to form a complete test system.

[0081] Each reactor was sealed and placed in a 35°C constant temperature water bath. The stirring speed was set to 200 rpm, and the reaction was continued for 12 hours to ensure that the system reached thermodynamic equilibrium or the reaction endpoint.

[0082] After the reaction was completed, 1 mL of suspension was taken from each reactor and an equal volume of 5% trichloroacetic acid was added to terminate the various enzymatic reactions. The mixture was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was used to quantitatively determine the final concentration of phosphocreatine in the system by high performance liquid chromatography. The molar conversion rate of the reaction was calculated based on the initial creatine concentration.

[0083] Table 4. Synthesis yield test data of creatine phosphate under different catalytic systems

[0084] According to Table 4 and Figure 4The data in (a) and (b) show that the introduction of a coenzyme regeneration network into the reaction system and the degree of removal of contaminating enzymes directly affect the final synthetic yield. In an in vitro single-enzyme catalytic environment, creatine kinase-catalyzed phosphorylation is a reversible process, and its thermodynamic equilibrium is unfavorable for the large accumulation of products in the aqueous phase. As the reaction proceeds, the concentration of the byproduct adenosine diphosphate (ATP) gradually increases in the system. This free molecule competes with ATP for enzyme binding sites, leading to product inhibition, and also accelerates the reverse reaction.

[0085] In Comparative Example 6, lacking a polyphosphokinase-assisted system, the system relied solely on the initial addition of 5 mmol / L adenosine triphosphate (ATP), resulting in a conversion rate of only 8.24%, indicating that the reaction reached thermodynamic equilibrium at a low product concentration. After introducing polyphosphokinase and sodium hexametaphosphate, the conversion rates in Examples 1 to 4 all exceeded 79%, reaching over 39 mmol / L. Polyphosphokinase utilizes sodium hexametaphosphate as a phosphate donor to rephosphorylate the generated adenosine diphosphate (ATP) to ATP. This in-situ regeneration mechanism maintains a low concentration of ATP in the system, thereby reducing product inhibition. The continuous replenishment of ATP alters the original chemical equilibrium, promoting the main reaction towards creatine phosphate synthesis. Although Comparative Example 5 was equipped with an auxiliary regeneration system, the crude enzyme solution used was not heat-treated for impurity removal, resulting in a conversion rate of 36.68%.

[0086] Residual adenosine triphosphate (ATP) hydrolases in host cells continuously degrade adenosine monophosphate (ATP) into adenosine monophosphate (ATP) or inorganic adenosine during the prolonged reaction, consuming the available adenosine substrate in the reaction system. With the loss of total adenosine, the regeneration system lacks reaction substrate, causing the main reaction to stop due to insufficient energy supply. The effective operation of the substrate regeneration system depends on the effective removal of background enzymes within the system; therefore, the thermal passivation process demonstrates the necessity of maintaining the stable operation of this catalytic network.

[0087] Test Example 5: This test case aims to evaluate the physical and mechanical strength and catalytic lifetime of polymeric composite supports constructed using a dual strategy of microenvironment charge modification and conformation locking under actual operating conditions. The specific experimental steps are as follows: Immobilized particles prepared in Example 1 with an initial total enzyme activity of 1000 U, conventionally embedded immobilized particles prepared in Comparative Example 2, and crude free creatine kinase enzyme solution prepared in Comparative Example 1 were used as test subjects.

[0088] 500 mL of standard test substrate solution was added to a jacketed, temperature-controlled, paddle-stirred reaction vessel. The substrate solution contained creatine at a concentration of 50 mmol / L, disodium adenosine triphosphate and magnesium sulfate heptahydrate at 5 mmol / L each, and sodium hexametaphosphate at a final concentration of 60 mmol / L. The pH of the system was maintained at 8.2. The test substances were then added to the reaction vessel, and the first batch of catalytic reactions was started under constant temperature of 35°C and stirring at 200 rpm for 4 hours.

[0089] After each batch reaction was completed, stirring was stopped and the catalyst was recovered and separated. For immobilized particles (Example 1 and Comparative Example 2), gravity filtration was used to intercept them using an industrial stainless steel standard sieve; for free enzymes (Comparative Example 1), a polyethersulfone tangential flow ultrafiltration membrane was used for concentration and retention.

[0090] The recovered catalyst was washed once with 50 mL of fresh buffer solution, and then an equal volume and concentration of fresh test substrate solution was added in situ to start the next catalytic cycle. This process was repeated for 20 consecutive batches.

[0091] Thirty minutes before the end of the 1st, 5th, 10th, 15th, and 20th batches of reaction, a suitable amount of sample solution was taken from each reaction vessel for quenching, and the reaction rate of phosphocreatine production in that batch was quantitatively determined by high-performance liquid chromatography. The initial reaction rate of the first batch was defined as 100%, and the relative enzyme activity retention rate of each test object in different batches was calculated.

[0092] Table 5. Relative enzyme activity retention rate test data of different catalytic systems under multiple batches of cyclic use.

[0093] According to Table 5 and Figure 5 The data shows that the batch stability of the catalytic system varies significantly under multiple batches of stirred reaction conditions. Although the free enzyme does not have carrier mass transfer resistance, it is difficult to maintain stable catalytic activity in multiple batches of reaction. After a small amount of recycling, the relative enzyme activity of the free creatine kinase in Comparative Example 1 had dropped to 17.3% in the fifth batch. The pump shear force generated by the ultrafiltration device when intercepting free proteins can easily lead to denaturation and inactivation of free proteins and cause membrane pore blockage, resulting in a significant loss of catalytic ability of the free enzyme after several cycles.

[0094] Comparative Example 2, employing a traditional direct encapsulation process, initially showed some matrix retention, but its enzyme activity retention rate declined to 43.8% in the 10th batch as the number of reaction cycles increased. The gelatin carrier was prone to swelling and structural disintegration under prolonged mechanical stirring in a slightly alkaline aqueous phase, leading to the loss of encapsulated enzyme molecules along with the carrier. Example 1, combining branched polyethyleneimine with a suitable concentration of glutaraldehyde for chemical cross-linking, maintained a relative catalytic activity of 77.4% even after 20 batches. The polymer modifier and gelatin molecular chains formed a composite polymer network under the action of the cross-linking agent, inhibiting excessive swelling of the gel and improving the mechanical stability of the particles. Pre-incubated enzyme molecules were embedded within the network, reducing physical shedding. This polymer network enabled the carrier to maintain structural integrity during multiple cycles, improving the catalyst's reusability.

Claims

1. A method for preparing phosphocreatine catalyzed by an immobilized enzyme, characterized in that, Includes the following steps: Dissolve 5-15 parts by weight of gelatin in 99-110 parts by weight of deionized water, add 0.05-1.5 parts by weight of positive charge modifier, mix well and cool down to obtain a blended carrier solution for constructing positively charged microregions. Add 0.1-0.5 parts by weight of creatine and 0.1-0.5 parts by weight of magnesium salt to 20-40 parts by weight of crude creatine kinase enzyme solution for pre-incubation to obtain a pre-incubation mixture. Mix the pre-incubation mixture with the co-mixed carrier solution to obtain a gel-forming mixture. The gelling mixture is dropped into a coagulation bath containing surfactant at a temperature of 3-5℃ and solidified into gel microspheres. The gel microspheres were placed in a buffer solution containing 0.2-1.0 parts by weight of crosslinking agent for crosslinking and washing to obtain immobilized creatine kinase particles; The immobilized creatine kinase particles, creatine, disodium adenosine triphosphate, magnesium salt, sodium hexametaphosphate, and polyphosphokinase coenzyme solution were added together to the reaction buffer to carry out the catalytic reaction. After the reaction was completed, the immobilized creatine kinase particles were separated and the reaction supernatant was collected to obtain phosphocreatine.

2. The method for preparing phosphocreatine by immobilized enzyme catalysis according to claim 1, characterized in that, The positive charge modifier is polyethyleneimine or chitosan oligosaccharide; The temperature control conditions for dissolving the gelatin in the deionized water are as follows: stir in a water bath at 50-60°C until dissolved, add the positive charge modifier and stir for 30-60 minutes, then lower the system temperature and maintain it at 35-40°C.

3. The method for preparing phosphocreatine by immobilized enzyme catalysis according to claim 1, characterized in that, The coagulation bath containing surfactant is composed of 490-510 parts by weight of Tris-HCl buffer solution with a pH of 8.0-8.5 for coagulation and 1-3 parts by weight of polyoxyethylene sorbitan monooleate. The gelling mixture is dripped into the coagulation bath containing the surfactant at a flow rate of 1.0-2.0 ml per minute using a micro-injection pump. After the dripping is completed, the gelling mixture is allowed to stand and age in the coagulation bath containing the surfactant for 30-60 minutes.

4. The method for preparing phosphocreatine by immobilized enzyme catalysis according to claim 1, characterized in that, The pre-incubation conditions are: slow stirring and incubation at 20-30°C for 10-30 minutes; The crosslinking conditions during the crosslinking washing process are crosslinking by stirring at 4°C for 2-6 hours.

5. The method for preparing phosphocreatine by immobilized enzyme catalysis according to claim 1, characterized in that, The proportions and conditions for adding reactants to the reaction buffer solution are as follows: Add 2.5-10 parts by weight of the creatine, 5-12 parts by weight of the disodium adenosine triphosphate, 2.5-6 parts by weight of the magnesium salt, 5-20 parts by weight of sodium hexametaphosphate, 10-30 parts by weight of polyphosphokinase coenzyme solution and 15-50 parts by weight of the immobilized creatine kinase particles. The reaction buffer is selected from one of Tris-HCl buffer, HEPES buffer, MOPS buffer or glycine-sodium hydroxide buffer; Adjust the pH of the reaction buffer to 8.0-8.

5. After all the reactants have been added, keep stirring at 20-50°C and react for 1-5 hours.

6. The method for preparing phosphocreatine by immobilized enzyme catalysis according to claim 1, characterized in that, The magnesium salt is magnesium sulfate heptahydrate, and the crosslinking agent is glutaraldehyde.

7. The method for preparing phosphocreatine by immobilized enzyme catalysis according to claim 1, characterized in that, The preparation process of the crude creatine kinase solution includes: Recombinant Escherichia coli containing the rabbit creatine kinase gene was inoculated into Luria-Bertani liquid medium and cultured. After inducing expression with an inducer, the wet cells were collected by centrifugation. The wet bacterial cells were suspended in a lysis buffer and then homogenized using a high-pressure homogenizer to obtain cell lysis buffer. The cell lysate was placed in a constant temperature water bath at 40-45℃ for 15-20 minutes to complete the thermal denaturation of endogenous enzymes in the host. The precipitate was then removed by centrifugation, and the crude enzyme supernatant was collected to obtain the crude creatine kinase enzyme solution after thermal passivation and impurity removal.

8. The method for preparing phosphocreatine by immobilized enzyme catalysis according to claim 7, characterized in that, The inducing agent is isopropyl-β-D-thiogalactoside; The process of cultivation and induction is as follows: The recombinant Escherichia coli was inoculated into the Luria-Bertani liquid medium and cultured at 30-37°C to the logarithmic phase. The culture temperature was then lowered to 16-30°C, and 0.05-0.2 parts by weight of the isopropyl-β-D-thiogalactoside was added. The culture was continued for 16-24 hours.

9. The method for preparing phosphocreatine by immobilized enzyme catalysis according to claim 7, characterized in that, The process of suspending the wet bacterial cells in the lysis buffer and breaking them down is as follows: 9-11 parts by weight of the wet bacterial cells were suspended in 50-100 parts by weight of Tris-HCl buffer solution with a pH of 7.5-8.0 and cyclically disrupted 2-4 times at a pressure of 600-1000 bar. The centrifugation conditions for removing precipitates are as follows: Centrifuge at 8000-12000 rpm for 10-20 minutes.

10. The method for preparing phosphocreatine by immobilized enzyme catalysis according to claim 1, characterized in that, The preparation process of the polyphosphokinase coenzyme solution includes: Recombinant Escherichia coli containing the Escherichia coli polyphosphoric acid kinase gene was inoculated into Luria-Bertani liquid medium and cultured at 30-37℃ to the logarithmic phase. 0.1-0.5 parts by weight of isopropyl-β-D-thiogalactoside was added as an inducer, and expression was induced at 16-30℃ for 10-24 hours. Subsequently, the wet polyphosphoric acid kinase cells were collected by centrifugation. The polyphosphokinase wet cells were suspended in Tris-HCl buffer for lysis and then broken up using a high-pressure homogenizer or an ultrasonic homogenizer to obtain polyphosphokinase cell lysis buffer. The polyphosphokinase cell lysate was centrifuged at 3-5°C and 8000-12000 rpm for 10-20 minutes to remove the precipitate, and the supernatant of the coenzyme was collected to obtain the polyphosphokinase coenzyme solution.

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